Changes in synaptic transmission and protein expression in the brains of adult offspring after prenatal inhibition of the kynurenine pathway

During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog

Foxp4 Is Dispensable for T Cell Development, but Required for Robust Recall Responses

<div><p>Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4⁺ and CD8⁺ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3⁺ T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells <em>in vitro</em>. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or <em>in vitro</em> effector T cell differentiation. <em>In vivo</em>, despite effective control of <em>Toxoplasma gondii</em> and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.</p> </div

Deletion of Foxp4 at the DP stage does not alter thymocyte development.

<p>A) DNA isolated from cHET and cKO thymocytes was amplified using primers to detect wild-type, floxed, and deleted allelic sequences. Band sizes and identities are indicated. Representative of ten experiments. B) RNA isolated from WT (C57BL/6) and cKO thymocytes was assessed for Foxp4 by real time PCR. ND = not detectable. Representative of three experiments. C) Total cellularity was assessed in both cHET and cKO thymi from four-week-old littermates. Each point represents a single mouse. Mean and standard deviation are indicated. D) Thymocytes were stained for CD4 and CD8 and analyzed by polychromatic flow. Contour plots shown are previously gated on live, singlet-gated cells, negative for myeloid lineage markers (CD11b, CD11C, CD19, B220). Gated frequencies are indicated. Representative of twelve cHET and twelve cKO mice. E) Absolute numbers of thymocyte populations were determined. Each point represents an individual mouse. F) Thymocytes were stained with CD4, CD8, CD5 and HSA. Cells are gated on populations as indicated at the top of each column and assessed for CD5, TCRβ, and CD44 expression. Solid histograms are from cHET and bold lines are from cKO mice. Representative of 12 cHET and 12 cKO mice.</p

T cell activation induces normal proliferation and effector T cell differentiation in the absence of Foxp4.

<p>A) C57BL/6 CD4⁺ T cells were isolated and plated in wells coated with 1 µg/mL anti-CD3 and 5 µg/mL soluble anti-CD28. Wells were harvested on the indicated days. RNA was isolated and cDNA was generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042273#s2" target="_blank">Materials and Methods</a>. Foxp4 relative to β-actin transcript levels were normalized to day 0. Representative of 2 experiments. B) CD4⁺ T cells were isolated from spleens of cHET and cKO mice, labeled with CFSE, and cultured four days in wells coated with indicated concentrations of anti-CD3 and 5 µg/mL soluble anti-CD28. Dilution of CFSE was assessed on day 4. Representative of 4 cHET and cKO mice. C) CD4⁺ T cells from cHET and cKO mice were stimulated overnight in wells coated with anti-CD3 plus soluble anti-CD28. Harvested cells were stained for CD69 and cytokine receptor IL-7R. Histograms are gated on CD4 or CD8 TCRβ⁺ cells. Representative of 2 experiments. D) cHET and cKO CD4⁺ T cells were cultured in wells coated with anti-CD3 plus soluble anti-CD28. After four days, cells were stained for expression of activation markers CD44 and CD62L. Representative of 4 cHET and 5 cKO mice.</p

Foxp4 deletion alters recall responses to <i>Toxoplasma gondii</i> in the spleen and brain.

<p>A and C) Splenocytes and BMNC from mice infected with <i>T. gondii</i> 40 days earlier were cultured with anti-CD3, soluble tachyzoite antigen (STAg), or left unstimulated for twenty-four hours. Supernatants were analyzed for levels of IFNγ by ELISA, and normalized to a standard curve. Representative of 3 independent experiments. B and D) Splenocytes and BMNC were stimulated for four hours with PMA and Ionomycin in the presence of Brefeldin A, and assessed for intracellular IL-2 and IFNγ. Contour plots are gated on CD4⁺ or CD8⁺CD3⁺ cells. Relative percentage is shown within each gate. Splenocytes are representative of 9 Cre^NEG, 12 cHET and 15 cKO. BMNC are representative of 5 Cre^NEG, 7 cHET, and 7 cKO mice. E) CD8-depleted splenocytes from cHET and cKO mice were cultured in Th0 (left) or Th1 (right) polarizing conditions. Cells were restimulated in the presence of monensin. Histograms are gated on CD4⁺ T cells. cHET (filled histogram); cKO (black solid line); unstimulated (dashed line). Representative of 6 experiments.</p

Foxp4 is not required for the generation of peripheral CD4 and CD8 T cells.

<p>A) Absolute numbers from spleens from four-week-old cHET and cKO mice, including total spleen cellularity (top panel), numbers of CD4⁺ T cells (middle panel) and CD8+ T cells (bottom panel). B–C) Splenocytes cells from 6–8 week old cHET and cKO mice were stained for expression of CD4, CD8 and CD44 and CD62L or CD25, CD69, TCRβ, and IL-7 receptor (CD127) for immunophenotyping by polychromatic flow cytometry on LSRII. Histograms are gated on live, singlet CD4⁺ T cells. Representative of 4 independent experiments. D) Splenic Foxp3⁺ nTreg cells were identified by flow cytometry based on expression of CD4, CD25 and intracellular staining for Foxp3 protein. Gated frequencies are indicated. Representative of 7 cHET and 7 cKO mice. E) Frequencies of Treg were calculated using the splenic cellularity and relative frequency. Each point represents an individual mouse. Mean and standard deviation are indicated. F) Naïve CD4 T cells from cHET or cKO mice were polarized for four days <i>in vitro</i> in the presence of IL-2 with (left) or without (right) TGFβ. Wells were harvested and cells were assessed for expression of CD25 and Foxp3. Plots are gated on live, CD4⁺ cells. Representative of 4 experiments.</p

Foxp4 deficient CD4 T cells exhibit reduced memory recall responses following LCMV infection.

<p>Foxp4^FLOX/FLOXCre^NEG, cHET and cKO mice were infected with the Armstrong strain of LCMV by intraperitoneal injection at day 0, allowed to clear infection, and to generate memory T cells. At day 30 post-infection spleens were harvested for analysis. A) Frequencies of I-A^b:gp61⁺ CD44^hi CD4 T cells at day 30. Representative of 3 Cre^NEG, 5 cHET and 5 cKO mice across two independent infections. B) Numbers of I-A^b:gp61⁺ CD44^hi CD4 T cells were calculated. C) Splenocytes harvested at day 30 were restimulated with gp61 peptide for four hours <i>in vitro</i>, in the presence of Brefeldin A. Cells were assessed for cell surface expression of CD4, CD8, CD44, and stained intracellularly for IFNγ, TNFα, and IL-2. Representative plots of 3 Cre^NEG, 5 cHET and 5 cKO across two independent infections. D) Compiled frequencies of cells producing IFNγ, TNFα, or IL-2 (left), or polyfunctional cells producing all three cytokines (right). Cre^NEG, filled circles; cHET, filled squares; cKO, filled triangles.</p

<i>Toxoplasma gondii</i> infection does not lead to wasting or lymphopenia in Foxp4 cKO mice.

<p>Eight-week-old Foxp4^FLOX/FLOXCre^NEG, cHET and cKO mice were infected with <i>T. gondii</i> by intraperitoneal injection of 20 Me49 cysts. A) Weights of mice were assessed longitudinally and plotted relative to the percentage of each respective mouse's starting weight at d0. Mean and s.d. from 9 Cre^NEG, 12 cHET, and 15 cKO mice. B) Brain tissue was harvested at day 40 post infection and parasite DNA quantitated using SYBR probes specific for <i>T. gondii</i>. Levels of <i>T. gondii</i> DNA were normalized to a standard curve. Each point represents an individual mouse. Mean and standard deviation are shown. Representative of 3 individual experiments. C) Spleens were harvested at 40 days post infection and total spleen cellularity was determined. Each point represents and individual mouse. Representative of 3 experiments.</p